The bacterium Escherichia coli has a single, circular chromosome that is replicated and segregated with great precision to daughter cells during cell division. Replication proceeds bi-directionally from a single origin and terminates on the opposite side of the chromosome. The reletive simplicity of this system and the limited number of cell components required for its propagation make it a model system for DNA replication and segregation in general. We have developed a P1 parS GFP-ParB system for localization by fluorescent microscopy of any desired locus on the E. coli chromosome. The technique works well in living cells and allows us to follow the fate of chromosomal sequences through several generations by time-lapse microscopy. In addition, we have used the technique, in combination with flow cytometry, to determine the spatial distributions of given loci at defined points in the cell cycle in a cell population. We currently have data for the dynamic behavior of multipleloci distributed around the chromosome in cells growing with a simple cell cycle at moderate growth rates. Our principle conclusions are as follows:1. Chromosome segregation is primarily accomplished while replication is still ongoing.2. The terminus is replicated at the cell center and the daughter termini of all cells remain attached there until cell division, at which time they rapidly segregate away from each other as the cell divides. The terminal foci are at the true cell center as the cell approaches division : the foci co-localize with a segment of the FtsZ ring. 3. The capture of the termini at the cell center is independent of the xerCD site-specific recombination system. It also appears to be independent of the C-terminal domain of FtsK, a protein implicated in DNA hadling at the cell center, although it is disrupted in many of the ftsK mutant cells due to aberrant or absent cell division events. 4. A terminus domain of 160kb., centered on the dif recombination site, segregates as a unit at cell division. Positions flanking this region segregate prior to cell division. 5. Origins of replication segregate fairly early in the cell cycle. On average, there is a delay of about 1/4 of a generation between origin initiation and segregation. However, the delay varies widely from cell to cell, with some origins segregating immediately after initiation. 6. Origins segregate from the cell center toward the poles and are free to move about for some time before becoming attached to the new cell centers. Daughter origins sometimes re-associate after initial segregation and dissociate again. Cohesion of origins, although it often occurs, is not a necessary or invariant feature of the cell cycle. 7. Positions around the chromosome that are intermediate between the terminal domain and the origin segregate before cell division, at times roughly corresponding to their map positions. There are some positions whose average segregation time appears earlier that predicted by an orderly segregation of markers timed by replication order. These may be regions that are handled in some special way by the segregation machinery. This year, we have put special emphasis on one component of the chromosome segregation machinery, the SeqA protein. SeqA is a protein that binds specifically to the sequence GATC when the adenine bases are hemi-methylated. This substrate is produced when chromosome loci are newly replicated. SeqA binds transiently to the newly replicated DNA that follows the progression of the replication forks. We have evidence that SeqA binding organizes and compacts the newly replicated DNA in a way that is important for proper DNA segregation. In collaboration with Wei Yang (NIDDK), and Alba Guarne (McMaster U.) we have solved the structure of much of the SeqA protein, and used the structural information to probe its function. We find that SeqA forms a helical filament on which the newly replicated DNA is wrapped. The filament grows from the replication fork by addition of dimers to the newly replicated GATC sequences. At the other end of the filament, dimers are lost as the DNA becomes fully methylated. Thus, a tract of the DNA progresses around the chromosome bound to a SeqA filament. This DNA organization appears to play a central role in segregation of the chromosome during replication. The overall mechanism for chromosome segregation in bacteria is clearly quite distinct from that of mitosis in higher organisms. However, there may be a strong resemblance between it and the mechanism that segregates the newly replicated DNA in eucaryotes into distinct sister chromatids.